Studies in this laboratory characterize the cellular pharmacokinetics of anticancer agents, in particular, the antifol methotrexate. This approach explores the properties of the membrane transport of cytotoxic agents as carefully distinguished from the subsequent interaction between the agent and its target site(s) within the intact cell. These studies have established a role for the component of intracellular methotrexate in excess of the tightly bound fraction as a critical determinant of inhibition of DNA, RNA and protein synthesis by this agent. These studies have suggested further that saturation of high-affinity binding sites with methotrexate does not alone result in the inhibition of dihydrofolate reduction to tetrahydrofolate while suppression of this metabolic pathway requires intracellular methotrexate in excess of the tightly bound fraction. These studies raise the possibility that forms of dihydrofolate reductase with a low affinity for methotrexate may be present within the cell and require for their suppression, appreciable levels of free drug within the intracellular compartment. Further studies will be undetaken to determine: (1) the effects of tightly bound and intracellular methotrexate in excess of this fraction on dihydrofolate reductase activity within the intact cell; (2) the characteristics of methotrexate transport at high extracellular drug levels - relevant to high-dose methotrexate-folinic acid rescue protocols and the effects of vinca alkaloids on the association of methotrexate with cells under these conditions; (3) the properties of folate and dihydrofolate transport and the characteristics of multiple transport routes for the folic acid group of compounds in mammalian cells. Other studies will be initiated to correlate the relationship between influx, exchangeable intracellular methotrexate levels, and drug efflux on cytotoxicity of methotrexate to a variety of murine leukemias of varying sensitivities to this agent to further assess the critical role of exchangeable intracellular methotrexate as a cytotoxic determinant.